Gene function in eukaryotes is dependent on the interaction of chromatin structure and DNA sequence-specific factors. The Polycomb repressor is a key chromatin-dependent regulator of developmental genes and cell differentiation in metazoans. As cell fates are determined, Polycomb repression is activated at specific DNA regulatory elements and expands across large chromatin domains. While many of these regulatory elements have been mapped, but what factors nucleate repression in some cell types, and how this spreads remains mysterious. My laboratory has developed novel methods to detect all occupied binding sites in a genome, based on the release of native protein-DNA particles by nuclease digestion. In this proposal, we use these methods to analyze the structure and composition of regulatory elements in different cell types in Drosophila, a model organism where Polycomb repression is best characterized. We will systematically analyze embryonic mesoderm to provide a comprehensive picture of the changes in regulatory elements and chromatin during development.